Parasitic infections have a long history of causing disease in humans and animals. These include protozoan parasites such as enteric and extra-intestinal amoebas, toxoplasmas and trichomonas. In addition, other human parasites include helminths such as roundworm, pinworm, hookworm, shisasomes and tapeworms. In general, diagnosis of intestinal parasites is confirmed by staining and microscopically identifying helminth eggs and larvae or protozoan trophozoites and/or cysts in fecal samples. Other assays, such as immunoassays, are also used in diagnosis.
Regardless of the method used, one problem in diagnosing parasitic infections is the delay between collecting and examining specimens, which, without a suitable fixative, results in rapid degradation of the specimen. Short of immediate processing, accurate diagnosis thus depends upon obtaining the best fixation of the specimen upon collection. Fixative solutions are therefore routinely used in processing specimens for parasitic diagnosis.
Fixatives used to preserve stool specimens, however, generally contain mercury, formaldehyde or formalin, which have a number of disadvantages that limit their use. For example, mercury-based fixatives, such as Schaudinn fixative with a mercuric chloride base, generally provide good definition, but present disposal concerns due to the prohibitive cost and the scarcity of disposal companies willing to handle mercury waste. Preservatives comprising formaldehyde or formalin raise similar environmental and health concerns.
In addition, formalin and mercury-based preservatives may limit the techniques that can be used to prepare and analyze the preserved specimens. For example, immunoassays utilizing fluorescent labels may become undetectable when formaldehyde is used and mercury based fixatives, such as Schaudinn's fixative, are not recommended for use in concentration techniques. (see Shimizu, R. Y. (Ed.) “Parasitology” in: Clinical Microbiology Procedures Handbook, 3rd ed. Wash. D.C., ASMPress, 2010. p. 9.2.2.3). This presents a problem in that concentration has become a routine procedure as a part of the complete ova and parasite examination and allows the detection of small numbers of organisms that may be missed by using only a direct wet smear (see Id., p. 9.3.4.1). Further, concentrated fecal sediment is recommended for the modified acid-fast and modified trichrome stains used for the coccidian and microsporidia, respectively (see Id.).
In addition, fixative compositions, even mercury-free fixatives, generally comprise polyvinyl alcohol (“PVA”). PVA is a plastic resin that may serve as a preservative and also serves as an adhesive for the stool material, allowing the stool to adhere to glass microscope slides. PVA, however, also has disadvantages when used in fixative solutions. For example, samples containing some organisms, such as Trichuris trichiura (eggs), Giardia lamblic (cysts) and Isospora belli (oocysts), do not concentrate well from PVA-treated specimens. Also, PVA is generally not compatible with iodine stains, used for direct wet mounts because iodine will cause PVA to coagulate. Further, PVA may cause distortion of ova and larvae morphology and is difficult to prepare in the laboratory (see e.g., Shimizu, R. Y. (Ed.) “Parasitology” in: Clinical Microbiology Procedures Handbook, 3rd ed. Wash. D.C., ASMPress, 2010. p. 9.2.2.4); Price, D. L. Comparison of Three Collection-Preservation Methods Jar Detection of Intestinal Parasites Clin. Microbiology, Vol. 14, no. 6 (December 1981), p. 656-660).
In addition, glutaraldehyde-based fixative solutions present challenges in terms of costs and storage. Glutaraldehyde is light sensitive and also generally must be stored at sub-zero temperatures, or is at least, refrigerated, to prevent it from polymerizing. Further, glutaraldehyde is generally incompatible with histochemical assays since fixation in glutaraldehyde causes loss of antigenicity. Loss of antigenicity is likely due to the formation of intra- and intermolecular cross-linkages and resulting changes in the tertiary structure of the involved protein (see e.g., McLean, I. W. and P. K. Nakane, Periodate-Lysine-Paraformaldehyde Fixative A new Fixative for Immunoelectron Microscopy, J. Histochem and Cytochem., Vol. 22, no. 12 (1974) pp. 1077-1083).
Although glyoxal aldehyde (1, 2-ethanedione)-based fixatives in some cases may permit selective control over cross-linking (see e.g., Dapson R W, Glyoxal Fixation: How it Works and Why it Only Occasionally Needs Antigen Retrieved. Biotech. Histochem., Vol. 82, no. 3 (January 2007), pp. 161-166), additional processing steps, suitable catalysts, reaction accelerators and/or high pH and high temperatures may be necessary to restore immunoreactivity. For example, glyoxal-based fixatives generally are not compatible with immunoassays for detecting Giardia and Cryptosporidum infection.
For the foregoing reasons, there is a need for a fixative suitable for fixing biological specimens which 1) do not contain mercury or formaldehyde; 2) are compatible with concentration procedures, permanent staining procedures, latex agglutination, direct and indirect immunofluorescence and enzyme immunoassays; 3) are compatible with different parasitic stages (e.g. cysts, eggs, larvae and trophozoites); and 4) are efficacious without the addition polyvinyl alcohol or the addition of aldehydes such as glyoxal and glutaraldehyde.